Tuberculosis is a massive problem in global public health. An estimated one-third of the world population is latently infected and active disease accounts for 8.6 million cases and 1.3 million deaths each year. The standard therapy involves a four-drug cocktail that requires six months of treatment and carries significant side effects. As a result, multidrug-resistant (MDR) and extensively drug-resistant (XDR) strains are growing in incidence. Thus, new antibiotics with novel mechanisms of action are urgently needed to combat tuberculosis. To address this problem, we have developed salicyl-AMS, the first-in-class inhibitor of siderophore biosynthesis in Mycobacterium tuberculosis. Siderophores are iron-chelating natural products that are produced by pathogenic bacteria and used to capture iron, an essential nutrient, from human host proteins. Siderophore biosynthesis and transport pathways have been genetically validated as potential therapeutic targets in vertebrate animal models of tuberculosis as well as other bacterial infections. In M. tuberculosis, mycobactin siderophores are synthesized by non-ribosomal peptide/polyketide synthetases encoded by the mbt gene cluster. The first committed step in mycobactin biosynthesis is catalyzed by MbtA, a salicylate adenylation enzyme that activates salicylic acid with ATP to form a tightly-bound salicyl-AMP intermediate, which then condenses with a thiol nucleophile on MbtB to form a salicyl thioester for further downstream processing. Salicyl-AMS is a stable, non-hydrolyzable mimic of the salicyl-AMP intermediate, and we have demonstrated that it is a low-nM, tight-binding inhibitor of MbtA; blocks mycobactin biosynthesis in cell culture; and potently and selectively inhibits M. tuberculosis growth under iron-deficient conditions that mimic the host environment. Moreover, we have recently demonstrated the first in vivo efficacy of salicyl-AMS in a mouse model of tuberculosis, providing critical pharmacological proof-of-concept for this novel antibiotic strategy. Salicyl-AMS now requires lead optimization to improve its pharmacological and toxicological properties. Herein, we propose to develop optimized salicyl-AMS analogues through a multidisciplinary collaboration between Derek Tan, Luis Quadri, and William Bishai, comprising combined expertise in organic synthesis, medicinal chemistry, biochemistry, microbiology, bacterial genetics, pharmacology, toxicology, and mouse models of tuberculosis. We will use a comprehensive approach involving rational chemical modifications to address pharmacological and toxicological limitations of salicyl-AMS. Extensive in vitro structure-activity relationships are already in hand and we have already developed analogues with improved pharmacological properties and also eliminated initially observed dose-limiting toxicity. Our overall goal is to develop one or more advanced leads with optimized pharmacological profiles and high in vivo antibacterial efficacy for further preclinical and ultimately clinical development. We will also use these compounds to probe of the effects of siderophore biosynthesis inhibition on bacterial growth and virulence in drug mechanism-relevant backgrounds.